Tuberculosis (TB) is a leading cause of preventable death. The ability of Mycobacterium tuberculosis (Mtb) to remain latent in a nonreplicating drug resistant state in the host for decades, to be reactivated when the immunity of the person weakens, is a major impediment to curing and eradicating this disease that causes nearly two million deaths annually. Fatty acids are known to be the major energy source required for the persistence of Mtb in the host. However, the source of the fatty acids remains unknown. We postulate that Mtb stores energy as triacylglycerol (TG) and wax esters (WE) to successfully go through latency. The steps Mtb uses to store and mobilize these energy sources could be ideal targets for novel antilatency drugs that will eliminate latent Mtb. To elucidate these steps we propose to: (1) Generate gene-disrupted mutants for tgs, fcr, fabp, fatp, cut and lip genes that may encode enzymes that could be involved in the storage and mobilization of lipids for successfully going through latency. (2) Examine the biochemical functions of tgs, lip, cut, and fcr genes in Mtb a) Determine the effect of disruption of tgs, lip, cut and fcr genes in the accumulation of lipids in Mtb under stress conditions that induce a dormancy-like state b) Elucidate the possible role of the lip and cut mutants on the mobilization of the stored lipids c) Examine the possible role of tgs, lip and cut genes in acyl transfer reactions in Mtb lipid metabolism. (3) a) Utilize a novel in vitro multiple stress Mtb culture model of dormancy to identify the tgs and fcr genes involved in the accumulation of TG and WE during development of dormancy. b) Develop a method to use the multiple stress in vitro model to test antilatency drug candidates. (4) a) Test whether Mtb goes into a dormant state in THP-1 derived macrophages (TDM) under hypoxia b) Determine whether the lipid bodies that accumulate in Mtb within lipid-loaded TDM originate from host storage lipids and identify Mtb genes involved in lipid accumulation c) Determine whether the lipid body accumulation by Mtb in TDM is associated with development of Rif resistance by investigating whether mutants defective in lipid accumulation fail to develop Rif resistance. Gene-disrupted mutants that fail to manifest dormancy traits in both in vitro models of dormancy will be tested for their ability to successfully go through dormancy and reactivation in a rabbit containment/dexamethasone reactivation model of TB latency. They will also be submitted to TARGET for testing in a hollow fiber artificial granuloma model of dormancy in mice and determine whether storage lipid accumulation is involved in this in vivo dormancy model. (5) Gene-disrupted mutants that fail to manifest dormancy traits in both in vitro models of dormancy will be tested for their ability to successfully go through dormancy and reactivation in a rabbit containment/dexamethasone reactivation model of TB latency. They will also be submitted to TARGET for testing in a hollow fiber artificial granuloma model of dormancy in mice and determine whether storage lipid accumulation is involved in this in vivo dormancy model. The expected results are likely to help identify targets for novel drugs that can eliminate latent Mtb.